Brazilian Journal of Otorhinolaryngology 2022;88(4):505---510

Brazilian Journal of

OTORHINOLARYNGOLOGY
www.bjorl.org

ORIGINAL ARTICLE

Role of VPAC1 and VPAC2 receptors in the etiology of

pregnancy rhinitis: an experimental study in rats
a,∗
Burak Ulkumen , Muhammet Burak Batir b , Burcu Artunc Ulkumen c
,
d
Halil Gursoy Pala , Seda Vatansever e,f , Sirri Cam g

a
Manisa Celal Bayar University, Faculty of Medicine, Department of Otorhinolaryngology-Head Neck Surgery, Manisa, Turkey
b
Manisa Celal Bayar University, Faculty of Science and Letters, Department of Biology, Manisa, Turkey
c
Manisa Celal Bayar University, Faculty of Medicine, Department of Obstetrics and Gynecology, Manisa, Turkey
d
The University of Health Sciences Tepecik Training and Research Hospital, Department of Obstetrics and Gynecology, İzmir,
Turkey
e
Manisa Celal Bayar University, Faculty of Medicine, Department of Histology-Embryology, Manisa, Turkey
f
Near East University, Experimental Research Center of Health (DESAM), Mersin, Turkey
g
Manisa Celal Bayar University, Faculty of Medicine, Departamento de Genética Médica, Manisa, Turkey

Received 13 January 2020; accepted 28 June 2020

Available online 1 August 2020

KEYWORDS
Abstract
Pregnancy rhinitis;
Introduction: Pregnancy rhinitis is a common sex hormone-related otorhinolaryngological dis-
VPAC1;
order. There are some epidemiological and physiological studies on pregnancy rhinitis, but
VPAC2;
histopathological and biomolecular changes have not been studied thoroughly.
Estradiol;
Objectives: The receptors VPAC1 and VPAC2 are known for their roles in allergic rhinitis. On
Progesterone
the other hand, activation of subclinical allergy has been suggested in the pathophysiology of
pregnancy rhinitis. Therefore, we aimed to compare the physiological and gestational pattern
of VPAC1 and VPAC2 expression in rat nasal mucosa.
Methods: Twenty adult Wister albino female rats were enrolled into the study. Two groups
constituted as 10 control (group A) and 10 pregnant (group B) rats. They were fed ad libitum and
sheltered at room temperature (22◦ ±2 ◦ C). The rats were sacrificed at the 20th day of gestation
by intraperitoneal injection of 400 mg/kg Na-pentobarbitone. Then, 10 − 15 mL of blood was
taken, and samples were reserved for the detection of serum estradiol and progesterone levels
by ELISA test. The nasal septum was resected and divided in half for immunohistochemical
analyses and real time polymerase chain reaction testing of VPAC1 and VPAC2.

∗ Corresponding author.
E-mail: drburak@gmail.com (B. Ulkumen).
Peer Review under the responsibility of Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial.

https://doi.org/10.1016/j.bjorl.2020.06.015
1808-8694/© 2020 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. This is an open
access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
 B. Ulkumen, M.B. Batir, B. Artunc Ulkumen et al.

Results: VPAC1 and VPAC2 were found to be in all layers of septal specimens, but the immunos-
taining of surface epithelium was more distinct in specimens of both groups. We demonstrated
higher overall staining intensity in the pregnant group. PCR revealed significant increase in
expression of VPAC1 (p = 0.023) and VPAC2 (p = 0.021) in pregnant group when compared with
control group. In addition, we demonstrated upregulatory effect of estradiol and progesterone
on the vasoactive intestinal peptide receptor expression.
Conclusions: Gestational up-regulation of nasal VPAC1 and VPAC2 was shown both by PCR and
immunohistochemical analysis. These findings support the hypothesis that PR is caused by the
activation of subclinical allergy that is present before pregnancy.
© 2020 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published
by Elsevier Editora Ltda. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

Introduction reveal a common pathway which would link up the phys-

Fluctuations in serum gonadocorticoid levels cause some
adverse Otorhinolaryngological (ORL) signs and symptoms.1,2
It is known that physiological, pathological or iatrogenic
conditions cause change in gonadocorticoid levels.3---5 Preg-
nancy, as a physiological condition, also induces some sex
hormone- related ORL symptoms.6,7 One of them is Preg-
nancy Rhinitis (PR), which is defined as the presence of nasal
congestion, rhinorrhea and sneezing which arise particularly
during gestation and subside in 3 weeks’ time after delivery.
In addition, history of allergy and other nasal pathologies are
defined as exclusion criteria for PR.3,8
PR leads to a predisposition to snoring and even obstruc-
tive sleep apnea syndrome, which induces some maternal
and fetal morbidities.9---11 The pathophysiology of PR is still
not fully understood. But increased serum levels of particu-
lar hormones (Progesterone [PG], Estradiole [E2], placental
growth hormone, human chorionic gonadotropin) have been
asserted as the triggering factor.10 Activation of subclinical
allergy has also been suggested as the pathophysiology of
PR by some studies.3,12---14 However, there is no high level
of evidence to support that Allergic Rhinitis (AR) and PR
share similar pathophysiological processes. In addition, the
impact of sex hormones on nasal mucosa has been studied in
various aspects.15---20 But there is very limited data regard-
ing histopathological and biomolecular changes in the nasal
mucosa of patients having PR.21,22
Vasoactive Intestinal Peptide (VIP) is a peptide hormone
which is known to be secreted in various tissues.23 VIP is
mainly known for its immunomodulatory functions.24 It takes
part in regulation of immune response through 4 receptors
(VPAC-1, VPAC-2, PAC1, CRTH2). In the upper respiratory
tract, VIP mainly functions via VPAC1 and VPAC2. On the
other hand, upregulation of nasal VPAC-1 and VPAC-2 expres-
sion was reported in AR.25 The link between these two
receptor proteins and allergy was also shown in some other
studies.24,26 In consideration of these findings we hypoth-
esize that the pattern of VPAC-1 and VPAC-2 expression
would significantly change in the nasal mucosa of preg-
nant rats when compared with non-pregnant ones. Discovery
of any gestational change in nasal expression of these 2
biomolecules may partly unveil the histopathological back-
ground of PR. Besides, we may have the opportunity to
iopathology of AR and PR. By this means new treatment
strategies could be proposed for PR.
As far as we know, the nasal expression of VPAC1 and
VPAC2 has not been studied in relation to pregnancy before.
In this study, we explored the physiological pattern of VPAC-
1 and VPAC-2 expression in rat nasal mucosa as well as the
impact of pregnancy on these receptors. Additionally, the
effect of PG and E2 on the expression of these 2 receptors
was also studied.
Methods
Animals
This animal experimental study was done in institutional
experimental animals’ research and application center in
accordance with the accepted policy on the use of animals.
Institutional Animal Ethics Committee approved the overall
procedures.
Twenty adults (8 − 12 week old) Wister albino female
rats were involved in the study. The rats were fed ad libi-
tum and sheltered at room temperature (22◦ ±2 ◦ C) on a
12 h light-dark cycle. All procedures were done during day-
time. Thirty-two female rats were housed together with
male rats in the ratio of 4/1 for overnight. The following
morning vaginal smears of female rats were examined for
sperm existence. We accept the observation of sperm as
the day 0 of the gestation as described before.27---29 The
first 10 rats with negative and 10 rats with positive vaginal
smear were chosen from among 32 rats as the control group
(Group A) and the pregnant group (Group B), respectively.
The remaining 12 rats were not enrolled into the study.
Gestation period of Wister albino rat has been reported as
approximately 22 (21 − 26) days.29 For this reason, we sac-
rificed the rats at 20thday of gestation by intraperitoneal
injection of 400 mg/kg Na-pentobarbiton.30 After the loss of
consciousness and righting reflex, 10 − 15 mL of blood was
taken by a 23 G needle.. Blood samples were reserved for
the detection of serum E2 and PG levels by ELISA.
We then shaved the nasal dorsum (Fig. 1a). We separated
the nasal bones from the maxilla in an upward manner and
revealed the nasal cavity macroscopically as described by
Alvites et al. (Fig. 1b).30 We then resected the cartilaginous

506
 Brazilian Journal of Otorhinolaryngology 2022;88(4):505---510

Figure 1 (a) External view of rat’s rhinarium (dorsal hair shaved), (b) Nasal roof (Os nazale) are mobilized in upward direction, (c)
Internal macroscopic view of both nasal cavities (Cavum nasi) after removal of nasal roof. The septum (Septum nasi osseum), middle
nasal chonca (Concha nasalis media) and frontal bone (Os frontale) are seen. Arrow: Os nazale; 1 and 2: Middle nasal conchae; 3:
Septum with respiratory epithelium; 4: Vomer with olfactory mucosa; * Frontal bone.

part of the septum, taking care not to damage the mucosal
integrity. The resected specimen was divided in half for
immunohistochemical analyses and real- time Polymerase
Chain Reaction testing (PCR).
Immunohistochemistry (IHC)
The pecimens were fixed by formaldehyde solution of
10% for 24 h. Next, dehydration and paraffin emplace-
ment were accomplished. Paraffin blocks were dissected at
a 4 ␮m thickness. For primary examination, Hematoxylin-
Eosin (H&E) dye was utilized. ‘‘VPAC1 Antibody (B-4):
sc-377152’’ and ‘‘VPAC2 Antibody (AS69): sc-52795’’ were
used in accordance with the recommended protocols for
immunohistochemical detection of VPAC1 and VPAC2 (Santa
Cruz Biotechnology, Inc., TX, USA). Concentration of VPAC1
and VPAC2 was 200 ␮g/mL and 50 ␮g/mL, respectively.
The control of immunospecifity of each group was per-
formed by the replacement of primary antibodies with
Phosphate Buffered Saline (PBS). Immunohistochemical pat-
tern of VPAC1 and VPAC2 expression was examined by light
microscopy (Olympus BX41). Immunostaining for VPAC1 and
VPAC2 was evaluated in terms of structural layers of nasal
mucosa.
ELISA
EDTA, sodium citrate and heparin were mixed with the blood
samples. Then the mixture was centrifuged at 3000 rpm for
10 min. The supernatant stored at −80 ◦ C. Rat E2 (estradiol)
ELISA Kit and General Progesterone (PG) ELISA Kit were used
for quantitative measurement of serum E2 and PG levels,
respectively (MyBioSource, Inc., CA, USA).29
RNA extraction and quantitative Real-Time PCR
(qRT-PCR) analyses
Total RNA extraction was carried out from the laryngeal
mucosa with using TRIzol® Reagent in the combination
with the PureLink® RNA Mini Kit (Thermo Fisher Scientific,
12183555). RNA expression levels of VPCA1 and VPCA2
were acquired from the extracted RNA samples with using
forward VPCA1F1 primer 5 -ATCAACTCCTCCCTGTGGTG-3 ,
reverse VPCA1R1 primer 5 -GGGCTGCTATCATTCTTCCC-
3 , forward VPCA2F1 5 -GGACAGTGTGCTCTACTCCA-3 ,
reverse VPCA2R1 5 -GCCAGTAGAAGTTCGCCATG-3 and
QuantiFast SYBR Green qRT-PCR Kit (Qiagen, 204154).
Separately prepared mixture of AQP5F1, AQP5R1, Quan-
tiFast SYBR Green and TREKF1, TREKR1, QuantiFast
SYBR Green was analyzed in the Rotor-Gene Q (Qia-
gen, Hilden, Germany). Normalising of the target genes
expression changes was performed according to the
␤-microtubulin (B2M) (B2MF1 5 -TCTCTCTTTCTGGCCTGGA-
3 , B2MR1 5 -TGTCGGATGGATGAAACCC-3 ) and
Hypoxanthine Phosphoribosyl Transferase (HPRT1)
(HPRT1F1 5 -CGTCTTGCTCGAGATGTGAT-3 , HPRT1R1
5 -TTCAGTGCTTTGATGTAATCCAG-3 ) housekeeping gene
expression values. The related forward and reverse primers
were synthesized by Metabion company (Germany). qRT-PCR
cycling conditions started with the reverse transcription
step of 50 ◦ C (10 min), followed by PCR step comprised of
an initial activation/denaturation stage of 95 ◦ C (5 min),
followed by 45 cycles of denaturation 95 ◦ C (20 s), combined
annealing/extension 61 ◦ C (50 s). The 2−CT method was
used to calculate the relative changes in gene expression.31
Statistical analyses
Relative VPAC1 and VPAC2 expressions were compared
between Group A and B. Distribution of data were eval-
uated by Shapiro-Wilk test and results were presented as
mean ± Standard Deviation (SD). Variables of Group A and
B were compared by independent samples t-test or Mann-
Whitney U Test according to the results of Shapiro-Wilk test.
The impact of serum E2 and PG levels on VPAC1 and VPAC2
was analyzed by Pearson correlation test. Statistical signif-
icance was defined as p < 0.05. The Statistical Package for
the Social Sciences (SPSS) Version 21.0 (IBM Corp.; Armonk,
NY, USA) was used for statistical calculations.

507
 B. Ulkumen, M.B. Batir, B. Artunc Ulkumen et al.
Figure 2 Septal cartilage with covering mucoperichondrium.
Increased cellularity and sub-epithelial oedema is notable in
pregnant group. (a) Control group (b) Pregnant group (H&E
stain) (Scale Bars = 50 ␮m and 10 ␮m).
Figure 3 VPAC1 immunohistochemistry for (a) Control group
and (b) Pregnant group. VPAC1 was found to be expressed in all
layers of septal mucoperichondrium. Additionally, it was more
intense in the surface epithelium of both groups. Overall stain-
ing intensity was more explicit in specimens of pregnant group.
(Scale Bars =50 ␮m and 10 ␮m).
Results
Immunohistochemistry
We observed pseudostratified cuboidal-columnar type of
epithelium in Hematoxylin and Eosin (H&E) sections of nasal
septal cartilage. In addition, increased superficial cellular-
ity and sub-epithelial edema was notable in Group B when
compared with the Group A (Fig. 2).
VPAC1 and VPAC2 were found to be located in all layers of
septal specimen (epithelium, basement membrane, lamina
propria, cartilage). But the staining intensity of epithelium
was more distinct in specimens of both group (Figs. 3 and 4).
Although we did not use an objective measure like ‘‘H-
Scoring’’ for comparison of groups, we roughly observed
higher overall staining intensity in samples of Group B when
compared with Group A (Fig. 3b, Fig. 4b).
PCR and ELISA
A total of 20 Wister albino female rats (10 control, 10
pregnant) were enrolled into the experiment. The mean
relative expression of mRNA coding for VPAC1 of Group A
and B was 0.029 ± 0.016 and 0.055 ± 0.026, respectively.
The mean relative expression of mRNA coding for VPAC2 of
Group A and B was 0.003 ± 0.001 and 0.007 ± 0.004, respec-
tively. The mean serum E2 levels of Group A and B was
21.85 ± 1.45 pg/mL and 73.59 ± 3.01 pg/mL, respectively.
508
Figure 4 VPAC2 immunohistochemistry for (a) Control group
and (b) Pregnant group. VPAC2 was found to be stained in all
layers of septal mucoperichondrium. Subepithelial hypercellu-
larity was remarkable in the pregnant group (b). Overall staining
intensity was more explicit in specimens of pregnant group.
(Scale Bars =50 ␮m and 10 ␮m).
The mean PG levels of Group A and B was 14.99 ± 1.96 ng/mL
and 32.80 ± 3.96 ng/mL, respectively (Table 1). PCR and
ELISA values were found to be abnormally distributed
(p < 0.05). For this reason, Mann-Whitney U Test was used for
comparison of relative biomolecule expressions and serum
sex hormone (E2, PG) levels between groups.
Both relative VPAC1 (p = 0.023) and VPAC2 (p = 0.021)
expression was found to be significantly higher in Group B
when compared with Group A. E2 and PG were also found
significantly higher in Group B when compared with Group A
(p < 0.001) (Table 1).
Discussion
PR is a relatively common sex hormone-related ORL disor-
der. Its incidence has been reported between 10%−40%.8,12
Unfortunately, the awareness of PR among patients and
even physicians is quite low. PR may cause some serious
comorbidities like maternal hypertension, preeclampsia,
low APGAR score and fetal growth retardation.10,11,32 There
are some epidemiological and physiological studies on
PR,8,33,34 but histopathological and biomolecular background
has not been studied thoroughly. Besides, there are very
limited numbers of studies regarding the treatment for
this quite common disorder.12,32,34,35 Suggestions for any
treatment modality necessitates comprehension of phys-
iopathology and histopathology. For this reason, in this
animal experimental study, we aimed to reveal histochem-
ical and biomolecular (VPAC1 and VPAC2) changes of nasal
mucosa during gestation.
VIP plays a key role in allergic diseases like AR and
asthma. It particularly functions via VPAC1 and VPAC2 in
the upper respiratory tract.23 Stimulation of these 2 recep-
tors leads secretion of pro- and anti-inflammatory mediators
like prostaglandins and leukotrienes. This ends up with
extravasation of inflammatory cells into the mucosa. Other
well-known functions of VIP are bronchodilation, vasodila-
tion and increased glandular secretion. In addition, recent
studies emphasize the neuroimmune interaction of VIP in
the context of allergic diseases.23,36 All these effects may
also play a role in the etiopathogenesis of PR. In fact, some
researchers have been asserted that PR is caused by aggrava-
tion of preexisting subclinical allergy.3,12,13 Toppozada et al.
 Brazilian Journal of Otorhinolaryngology 2022;88(4):505---510
Table 1 Expression of VPAC1 and VPAC2 in nasal mucosa of rat, serum E2 and PG levels based on groups.
Biomolecules & sex hormones Control (Group A)
Biomolecules
VPAC1 (REV) 0.029 ± 0.016
VPAC2 (REV) 0.003 ± 0.001
Serum sex hormone levels
Estradiol (pg/mL) 21.85 ± 1.45pg
Progesterone (ng/mL) 14,99 ± 1.96 ng/mL
REV, Relative expression value.
a p-values obtained by Mann-Whitney U Test.
revealed some changes like AR in specimens of PR patients
by electron microscopy.13 On the other hand, Ellegard et al.
revealed increased levels of IgE against house dust mite in
PR patients.14 In this study, we revealed a statistically sig-
nificant increase of nasal VPAC1 (p = 0.023) and VPAC2 (p =
0.021) expression in pregnant rats when compared with the
non-pregnant ones (Table 1). This finding supports the argu-
ment of previous researchers.13,14 In other words, this study
supports the hypothesis that PR and AR may share a common
pathway for VIP.
Impact of sex hormones on the nasal mucosa has
been shown by previous researchers in various physiologi-
cal, iatrogenic or pathological conditions.3,5---7,13,15---22 Almost
all concluded that E2 and PG causes obstruction and
edema in the nasal cavity which is the case in this study
(Fig. 2b).3,6,13,15,16,21 In addition to these findings, we found
a relatively increased superficial higher cellularity in the
epithelial layer in samples of Group B. This may be a sign
of increased metabolism or activity. On the other hand,
very few researchers evaluate the underlying histochemi-
cal and biomolecular changes. Konno et al. revealed that
E2 up-regulates cholinergic muscarinic receptors while PG
down-regulates ␣1 -adrenergic receptors.20 These findings of
Konno et al. are compatible with the study of Fisher et al. in
relation to neuroimmunomodulation.36 Fisher et al. revealed
higher VIP contents in mucosal nerve fibers of AR patients
when compared with control. Our findings also support this
phenomenon. The higher nasal VPAC1 and VPAC2 expression
in pregnancy that was revealed in this study can also be the
triggering factor for PR. Besides, Philpott et al. discovered
a positive correlation between oestrogen-␤ (ER␤) receptor
expression and rhinitis symptoms.17 Our findings also sup-
port this correlation. We revealed up-regulatory effect of E2
and PG on VPAC1 by PCR. But VPAC2 was found to be stim-
ulated only by E2. These findings may be the missing link
of the VIP pathway triggered by sex hormones, resulting in
extravasation of inflammatory cells into the nasal mucosa.
In the current study, both VPAC1 and VPAC2 were found
to be located in all layers of septal mucoperichondrium.
However, the surface epithelium of both groups demon-
strated higher immunostaining for both VIP receptors. In
addition, we revealed higher overall staining intensity in
samples of pregnant rats (Figs. 3 and 4). These IHC findings
are similar to the study of Kim et al.25 They demonstrated
increased staining intensity for VIP receptors in nasal mucosa
of AR patients. Therefore, we can suggest that PR and AR
have a similar effect on VIP receptors. On the other hand,
this strong immunostaining particularly demonstrated in the
509
Pregnant (Group B) p-valuea
0.055 ± 0.026 p = 0.023
0.007 ± 0.004 p = 0.021
73.59 ± 3.01 pg/mL p<0.001
32.80 ± 3.96 ng/mL p<0.001
surface epithelium can be interpreted as a sign of hyper
responsiveness of nasal mucosa in pregnancy.
Conclusions
We revealed gestational upregulation of VPAC1 and VPAC2
expression in rat nasal mucosa for the first time both by
PCR and IHC. The current study supports the hypothesis
that PR is caused by the activation of a subclinical allergy
that is present before pregnancy. Further studies are needed
to unveil the complete histopathological and biomolecular
changes regarding PR.
Ethical approval
This animal experimental research was approved by the
Laboratory Animals Local Ethics Committee of Manisa Celal
Bayar University (28.04.2015/77.637.435-29) and was per-
formed in institutional Experimental Animals Research and
Application Center in accordance with the accepted policy
on the use of animals.
Committee Name: The Laboratory Animals Local Ethics
Committee of Manisa Celal Bayar University. Permit number:
(28.04.2015/77.637.435-29).
Conflicts of interest
The authors declare no conflicts of interest.
Funding
Unit of Scientific Research Projects (BAP) of Manisa Celal
Bayar University (Grant number: 2014-131).
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